Liquid-Liquid Phase Separation in Hemoglobins: Distinct Aggregation Mechanisms of the β6 Mutants

Abstract

Reversible liquid-liquid (L-L) phase separation in the form of high concentration hemoglobin (Hb) solution droplets is favored in an equilibrium with a low-concentration Hb solution when induced by inositol-hexaphosphate in the presence of polyethylene glycol 4000 at pH 6.35 HEPES (50 mM). The L-L phase separation of Hb serves as a model to elucidate intermolecular interactions that may give rise to accelerated nucleation kinetics of liganded HbC (beta6 Lys) compared to HbS (beta6 Val) and HbA (beta6 Glu). Under conditions of low pH (pH 6.35) in the presence of inositol-hexaphosphate, COHb assumes an altered R-state. The phase lines for the three Hb variants in concentration and temperature coordinates indicate that liganded HbC exhibits a stronger net intermolecular attraction with a longer range than liganded HbS and HbA. Over time, L-L phase separation gives rise to amorphous aggregation and subsequent formation of crystals of different kinetics and habits, unique to the individual Hb. The composite of R- and T-like solution aggregation behavior indicates that this is a conformationally driven event. These results indicate that specific contact sites, thermodynamics, and kinetics all play a role in L-L phase separation and differ for the beta6 mutant hemoglobins compared to HbA. In addition, the dense liquid droplet interface or aggregate interface noticeably participates in crystal nucleation.

Soft matter perspective on protein crystal assembly

“…Although each of these steps presents several experimental challenges, crystallization is by far the most troublesome [9][10][11]15]. Under standard biological conditions, most proteins do not easily crystallize.…”

“…Some proteins are characterized by a decreasing protein solubility with increasing temperature, i.e., an inverted solubility [158][159][160]. Sometimes a single mutation can flip the solubility curve [56,108] or significantly change the assembly kinetics [15,161]. This phenomenon is tentatively attributed to the temperature dependence of the water entropy [162,163], but remains poorly understood overall.…”

Dense liquid droplets as a step source for the crystallization of lumazine synthase

“…The dense liquids seen with the proteins lysozyme, g-crystalline, hemoglobin A, C, and S [4,8,[38][39][40], and others are metastable with respect to an ordered solid phase of the respective proteins, but, very importantly, are stable, i.e., have lower free energy than the initial low-density solution. After small droplets of such dense liquids form, their lifetime is limited by two events: decay into the initial solution, or transformation into an ordered solid phase.…”

Nucleation of protein condensed phases

“…This dependence indicates that the condensed phases are favored at a low temperature. Opposite examples, where crystallization or liquid-liquid separation are favored at high temperature, exist: hemoglobin C has the so-called retrograde temperature dependence of the solubility, whereby the solubility increases at a low temperature (Feeling -Taylor et al 1999; dense liquid appears in solutions of several hemoglobin variants upon temperature increase , Chen et al 2004.…”